Mammalian cells have evolved multiple sensing pathways to detect foreign invasion. Recent evidence[unreadable] indicated that several proteins previously implicated in apoptosis also participate in innate immunity[unreadable] through Toll-Like receptors (TLR)-dependent and independent pathways. These include TRAIL-R, an[unreadable] apoptosis-inducing member of the tumor necrosis factor receptor family and FADD, a death-domain[unreadable] containing adapter protein. TRAIL can be induced through TLR-> interferon pathway. Subsequent[unreadable] activation of TRAIL-R by TRAIL results in negative feedback loop of NF-kappaB transcription factor. TRAIL-R-/-[unreadable] dendritic cells/macrophages stimulated with TLR-3/4 ligands display enhanced cytokine levels and loss of[unreadable] NF-kappaB homeostatic regulation. FADD, reminiscent of the Drosophila Imd-FADD innate immune response[unreadable] system, was found to be crucial for intra-cellular dsRNA-activated gene expression in human/mouse. In[unreadable] the presence of interferon, FADD-/- fibroblasts were not able to clear RNA viruses that include Influenza.[unreadable] Thus, FADD is part of an alternative TLR-independent mammalian pathogen-sensing pathway. In this[unreadable] application, we hypothesize that TRAIL-R and FADD play significant but distinct roles in the[unreadable] innate immune responses against a variety of viruses and selected parasite. In Aim 1, viruses from[unreadable] various families, including several in the bio-defense category like Influenza, Vaccinia and LCMV (with[unreadable] Project 3) and the intracellular parasite Toxoplasma gondii (with Project 1), will be used to determine the[unreadable] role of FADD and TRAIL-R in regulating host responses. We will first examine FADD-/- and TRAIL-R-/-[unreadable] fibroblasts for their ability to support viral replication. Microarray analysis will then be done to assess[unreadable] altered global gene expression, if any, in these cells. Dendritic cells/macrophage-specific FADD-/- mice will[unreadable] be generated. The host responses of these and TRAIL-R-/- mice against selected pathogens (Influenza,[unreadable] Toxoplasma, Cytomegalovirus) will be examined. Two-photon imaging studies in fluorescent transgenic[unreadable] mice in either TRAIL-R-/- or FADD tissue-specific deficient alleles will be used to assess host-pathogen[unreadable] interaction. In Aim 2, the signal transduction pathway leading to negative regulation of NF-KB by TRAIL[unreadable] will be examined. Signaling proteins involved in FADD-mediated innate immunity will also be identified.[unreadable] Microarray analysis will be performed to examine gene expression profile of TRAIL-R-/- macrophages.[unreadable] Mass spectrometry will then be used to identify TRAIL-R- or FADD-associated proteins in experiments[unreadable] involving Fas/TRAIL-R chimeric protein, tandem-affinity-protein technology and co-immunoprecipitation.[unreadable] Finally, RNAi knockdown approach will be used to assess the functional significance of any newly[unreadable] identified TRAIL-R or FADD associated proteins in innate immunity against selected bio-defense[unreadable] organisms.